bioRxiv preprint doi: https://doi.org/10.1101/536136; this version posted January 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Acquaviva et al., 30 Jan, 2019 – bioRxiv preprint v1 Ensuring meiotic DNA break formation in the mouse pseudoautosomal region Laurent Acquaviva1*, Michiel Boekhout1†, Mehmet E. Karasu1,2, Kevin Brick3, Florencia Pratto3, Megan van Overbeek1‡, Liisa Kauppi1§, R. Daniel Camerini-Otero3, Maria Jasin2,4* and Scott Keeney1,2,5* 1 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 2 Louis V. Gerstner, Jr., Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 3 Genetics & Biochemistry Branch, NIDDK, NIH, Bethesda, MD, USA. 4 Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 5 Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. * Correspondence to [email protected], [email protected] or [email protected] Current addresses: †UMC Utrecht, Oncode Insitute, Utrecht University, Utrecht, Netherlands; ‡Caribou Biosciences, Inc., Berkeley, CA, USA; §Faculty of Medicine, University of Helsinki, Helsinki, Finland. Sex chromosomes in males share only a diminutive homologous Results segment, the pseudoautosomal region (PAR), wherein meiotic double-strand breaks (DSBs), pairing, and crossing over must occur A distinctive PAR ultrastructure rich in pro-DSB factors for correct segregation. How cells ensure PAR recombination is We applied a cytogenetic approach to investigate the mouse PAR unknown. Here we delineate cis- and trans-acting factors that control structure in the C57BL/6J strain (B6). In most of the genome, axes PAR ultrastructure and make the PAR the hottest area of DSB elongate and DSBs begin to form during leptonema, then homologous formation in the male mouse genome. Prior to DSB formation, PAR chromosomes pair and axes are juxtaposed by the transverse filament chromosome axes elongate, sister chromatids separate, and DSB- protein SYCP1 (forming the synaptonemal complex, or SC) during promoting factors hyperaccumulate. These phenomena are linked to zygonema. Synapsis and recombination are completed during mo-2 minisatellite arrays and require ANKRD31 protein. We pachynema, then the SC disassembles during diplonema with homologs propose that the repetitive PAR sequence confers unique chromatin remaining attached at sites of crossovers (chiasmata) until anaphase I (1, and higher order structures crucial for DSB formation, X–Y pairing, 27). X and Y usually pair late, with PARs paired in less than 20% of and recombination. Our findings establish a mechanistic paradigm spermatocytes at late zygonema when nearly all autosomes are paired (7, of mammalian sex chromosome segregation during spermatogenesis. 8). At this stage, we found by conventional immunofluorescence microscopy that unsynapsed PAR axial elements (SYCP2/3 staining) Introduction appeared thickened relative to other unsynapsed axes and had bright HORMAD1/2 staining (Fig. 1A and fig. S1A,B) (28). Meiotic recombination forms connections between homologous We found that the PAR was highly enriched for DSB-promoting chromosomes that ensure accurate segregation (1). In many species, factors REC114, MEI4, MEI1, and IHO1 [proteins essential for genome- every chromosome must recombine, so a crucial challenge is to ensure wide DSB formation (22-24, 29)] as well as ANKRD31, a REC114 that every chromosome pair acquires at least one SPO11-generated DSB partner essential for PAR DSB formation (30, 31). All five proteins to initiate recombination (2). (hereafter RMMAI for simplicity) colocalized in several bright, irregular This challenge is especially acute in most male placental mammals “blobs” for most of prophase I (Fig. 1A–C and fig. S1C). Two blobs for sex chromosomes (X and Y), on which a DSB can only support were on the X and Y PARs as judged by chromosome morphology at late recombination if it occurs in the tiny PAR (3-7). The PAR in laboratory zygonema (Fig. 1A) and particularly bright fluorescence in situ mice is the shortest thus far mapped in mammals, at ~700 kb (5, 6). Since hybridization (FISH) with a probe for the PAR boundary (PARb) (Fig. only one DSB is formed per ten megabases on average in the mouse, the 1C). Other blobs highlighted the distal ends of specific autosomes (Fig. PAR would risk frequent recombination failure if it behaved like a typical 1C), which we revisit below. Undefined blobs are also apparent in autosomal segment (7, 8). However, the PAR is not typical, having published micrographs but were not explored further (21-24). Consistent disproportionately frequent DSB formation and recombination (4, 7, 9, with and extending other studies (21-24, 30, 31), all five proteins also 10). The mechanisms promoting such frequent DSBs are not known in colocalized in numerous small foci along unsynapsed chromosome axes any species. (Fig. 1B and fig. S1C), but PAR staining was much brighter. ANKRD31, Higher order chromosome structure plays an important role in MEI1 and REC114 enrichment on the PAR was already detectable in pre- meiotic recombination. DSBs arise concomitantly with development of leptotene cells during premeiotic S phase (fig. S1D), as shown for MEI4 linear axial structures that anchor arrays of chromatin loops within which and IHO1 (21, 23). DSBs occur (1, 11-13). The axis begins to form between sister Structured illumination microscopy (SIM) resolved the thickened chromatids during pre-meiotic replication (pre-leptonema) and includes PAR axes as two strands of axial core (Fig. 1D and fig. S2A,B) that were SYCP2 and SYCP3 (14, 15), cohesin complexes (16), and HORMA heavily decorated along their lengths with RMMAI proteins (Fig. 1E). domain proteins (HORMAD1 and HORMAD2) (17-20). Axes are also At the zygotene–pachytene transition, X and Y pair and initiate synapsis, assembly sites for IHO1, MEI4, and REC114 complexes (13, 21-24), then SC spreads bidirectionally—homologously in the PAR and non- whose functions as essential promoters of SPO11 activity are homologously between the non-PAR axes, which definitively marks incompletely understood (25, 26). early pachynema in mice (28, 32, 33) (Fig. 1D). Separation of PAR axes We previously showed that PAR chromatin is organized into short was readily observed by SIM in late zygonema before X and Y pairing loops on a long axis, (7). However, only a low-resolution view of PAR and synapsis, remained apparent after synapsis, then disappeared during structure was available and the cis- and trans-acting factors controlling early pachynema (Fig. 1D). A multi-core structure was also seen in PAR structure and DSB formation have remained largely unknown. earlier electron microscopy studies, but was staged incorrectly as occurring at mid-to-late pachynema (34) (fig. S2C,D). 1 bioRxiv preprint doi: https://doi.org/10.1101/536136; this version posted January 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Sex chromosome recombination In principle, the sister chromatid axes could be split apart (Fig. 1F) and the association of compact PARb chromatin with a long axis. or the PAR could be folded back on itself in a crozier configuration (fig. S2E). However, a crozier was ruled out by using SIM to define the path RMMAI proteins accumulate at mo-2 minisatellites of the PAR: the telomere binding protein TRF1 (35) decorated only the What are the cis-acting determinants of this PAR behavior? We tip of the chromosome (Fig. 1G) and the PARb probe yielded FISH deduced that DNA sequences shared between the PAR and certain signals symmetrically arrayed at similar positions on both axial cores autosomes might be responsible for recruiting RMMAI proteins because (fig. S2F). We conclude that each axial core is one of the sister the blobs consistently decorated the distal tips of specific autosomes that chromatids, with a “bubble” opened from near the PAR boundary almost also hybridized, albeit more weakly, to the PARb FISH probe (Fig. 1C). to the telomere (Fig. 1F). Axis splitting and REC114 enrichment Self-aligning the PARb probe sequence illustrated its highly occurred in the absence of SPO11 (Fig. 1H), thus neither property of the repetitive nature, including a central ~20-kb tandem array of a PAR is provoked by DSB formation. minisatellite called mo-2, with a 31-bp GC-rich repeat (36, 37) (Fig. 3A). These findings establish that the X and Y PARs adopt an elaborate BLAST searches detected additional mo-2 clusters at the non- axial structure that forms prior to homologous pairing and synapsis and centromeric ends of chr9 and chr13 in the mm10 genome assembly and disappears during early pachynema. It normally occurs at or before the chr4 from the Celera assembly (Fig. 3A), matching the reported time when most cells make PAR DSBs (which is in late zygonema or distribution (36, 37). Although these autosomal clusters are only 2 to 8 around the zygotene–pachytene transition (7)) but also forms in the kb in the assemblies, their copy number is likely underrepresented absence of DSBs and is preceded by accumulation of high levels of because they lie adjacent to gaps. RMMAI proteins, which starts in pre-leptonema. To test if RMMAI blobs correspond to mo-2 arrays, we used an oligonucleotide FISH probe with the mo-2 consensus sequence. This Dynamic remodeling of the PAR loop–axis structure probe gave a compact signal along the PAR axes that was similar to the To investigate temporal patterns of axis differentiation, RMMAI RMMAI blob pattern in shape and proportional intensity (Fig. 3B and composition, and chromatin loop configuration, we used SIM to compare fig.